Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to a protease which has at least 75% identity to a protease derived from Thermoascus aurantiacus, the amino acid sequence of which is shown in the appended sequence listing as amino acids 1 to 177 of SEQ ID NO: 2, and comprises at least one modification as compared to this protease (i.e., is a variant thereof).
• the invention also relates to DNA encoding these proteases, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of their production, as well as the use thereof, e.g. in production of fermentation products, e.g., ethanol.
• Proteases are well-known enzymes, as are the advantages of applying them in production of fermentation products. Proteases have been isolated from various sources, including a number of fungal and bacterial strains.
• protease activity protease activity
• polynucleotides encoding the polypeptides it is also an object of the invention to provide protease of improved properties as compared to the parent protease from which they were derived.
• the protease variants of the invention exhibit improved thermostability compared to the wild type parent protease.
• WO 2003/048353 discloses the wildtype protease of Thermoascus aurantiacus CGMCC No. 0670.
• the present invention relates to a method of producing a protease variant having protease activity and an improved thermostablity when compared to the wild-type parent protease, said method comprising culturing a cell into which has been introduced an expression vector comprising the following operably linked elements:
• a polynucleotide molecule encoding a protease variant which has at least 75% identity to the protease shown in amino acids 1 to 177 of SEQ ID NO: 2 and which comprises at least one modification as compared to amino acids 1 to 177 of SEQ ID NO: 2 in at least one position selected from the following: 27, 79, 82, 87, 1 12, 142, 2, 5, 6, 8, 26, 41 , 43, 46, 49, 53, 54, 73, 88, 104, 1 14, 1 15, 1 16, 126, 152, 157, 158, and 173,
• polynucleotide molecule is prepared by introducing at least one mutation into a DNA molecule encoding a protease
• said cell expresses the protease variant encoded by the polynucleotide molecule; and recovering the protease variant.
• the present invention relates to a protease which has at least 75% identity to amino acids 1 to 177 of SEQ ID NO: 2 and which comprises at least one modification as compared to amino acids 1 to 177 of SEQ ID NO: 2 in at least one position selected from the following: 27, 79, 82, 87, 1 12, 142, 2, 5, 6, 8, 26, 41 , 43, 46, 49, 53, 54, 73, 88, 104, 1 14, 1 15, 1 16, 126, 152, 157, 158, and 173 .
• the invention also relates to a nucleic acid sequence encoding these proteases, a nucleic acid construct comprising the nucleic acid sequence, an expression vectors comprising the nucleic acid construct, a host cell comprising the expression vector and/or the nucleic acid construct.
• the invention also relates to the use of the proteases, e.g. in a starch liquefaction, saccharirfication and/or fermentation process.
• the present invention relates to isolated variants of a parent protease, comprising a modification in at least one position corresponding to positions 27, 79, 82, 87, 1 12, 142, 2, 5, 6, 8, 26, 41 , 43, 46, 49, 53, 54, 73, 88, 104, 1 14, 1 15, 1 16, 126, 152, 157, 158, and 173 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has protease activity.
• the variants according to the invention have improved thermo-satbility.
• Protease activity means a polypeptide of EC. 3.4.-.-. In a preferred embodiment it is a polypeptide belonging to the EC 3.4.24 metalloendopeptidases. Protease activity may be determined according to the procedure described in Example 1 , e.g. using azo-casein assay or endo-protease assay using protazyme OL.
• the ENZYME site at the internet is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A.
• variant means a polypeptide having protease activity comprising at least one modification, i.e., a substitution, insertion, and/or deletion, compared to the amino acid sequence shown as 1 to 177 of SEQ ID NO: 2.
• at least one e.g. modification
• at least one means one or more, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications; or 12, 14, 15, 16, 18, 20, 22, 24, 25, 28, or 30 modifications; and so on, up to a maximum number of modifications of 44.
• the protease variants of the invention still have to be at least 75% identical to amino acids 1 to 177 of SEQ ID NO: 2.
• the variant has at least 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to amino acids 1 to 177 of SEQ ID NO: 2.
• the degree of identity is at least 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%, 99.1 %, 99.2%, 99.3%, or at least 99.4%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO: 2.
• Mutant means a polynucleotide encoding a variant.
• Wild-Type Enzyme means a protease expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.
• Parent or Parent Protease means a protease to which an alteration is made to produce the enzyme variants of the present invention.
• the parent may be a naturally occurring (wild-type) polypeptide or a variant thereof.
• Parental variant means that the starting point for making a variant protease was itself a variant compared to the wild type protease.
• Isolated variant means a variant that is modified by the hand of man.
• the variant is at least 1 % pure, e.g. , at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE.
• substantially pure variant means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1 %, and at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated.
• the variant is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation.
• the variants of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant by well known recombinant methods or by classical purification methods.
• Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
• the mature polypeptide is amino acids 1 to 177 of SEQ ID NO: 2.
• the signal peptide part can be predicted by programs known in the art (SignalP (Nielsen et al., 1997, Protein Engineering 10: 1 -6)). Amino acids 1 to 177 of SEQ ID NO: 2 is the expected mature part.
• the first amino acid of the mature part of an enzyme can be determined by N-terminal sequencing of the purified enzyme. The N- terminal of the mature polypeptide was confirmed by N-terminal sequencing. Any difference between the signal peptide part and the mature part must then be due to to the presence of a propeptide.
• Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having protease activity.
• the mature polypeptide coding sequence is nucleotides 535 to 1065 of SEQ ID NO: 1 based on SignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6) that predicts nucleotides 535 to 1065 of SEQ ID NO: 1 encode a signal peptide.
• Sequence Identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
• sequence identity the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.
• the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
• the output of Needle labeled "longest identity" is used as the percent identity and is calculated as follows:
• the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later.
• the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
• the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
• Isolated polynucleotide means a polynucleotide that is modified by the hand of man.
• the isolated polynucleotide is at least 1 % pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by agarose electrophoresis.
• the polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
• substantially pure polynucleotide means a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered polypeptide production systems.
• a substantially pure polynucleotide contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1 %, and at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated.
• a substantially pure polynucleotide may, however, include naturally occurring 5'- and 3'- untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, e.g., at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, and at least 99.5% pure by weight.
• the polynucleotides of the present invention are preferably in a substantially pure form.
• Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of its polypeptide product.
• the boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA.
• the coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.
• nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic.
• nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
• Control sequences means all components necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native or foreign to the polynucleotide encoding the variant or native or foreign to each other.
• control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
• the control sequences include a promoter, and transcriptional and translational stop signals.
• the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.
• operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
• expression includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
• Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to additional nucleotides that provide for its expression.
• host cell means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
• host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
• Improved property means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, thermal activity, thermostability, pH activity, pH stability, substrate/cofactor specificity, improved surface properties, product specificity, increased stability or solubility in the presence of pretreated biomass, improved stability under storage conditions, and chemical stability.
• Improved thermostability means a variant displaying retention of protease activity after a period of incubation at elevated temperature relative to the parent, either in a buffer or under conditions such as those which exist during product storage/transport or conditions similar to those that exist during industrial use of the variant. Whether or not a variant protease of the invention has an improved thermostability as compared to a parent protease may be determined as described in Example 1.
• the variant protease of the invention may have an improved thermostability as compared to a parent protease, wherein the improved thermostability is determined as increased relative activity.
• the variant protease of the invention may have an improved thermostability as compared to a parent protease, wherein the improved thermostability is determined as increased remaining activity.
• thermostability of the variant having protease activity is at least 1.05-fold, e.g., at least 1 .1-fold, at least 1.5-fold, at least 1 .8-fold, at least 2-fold, at least 5-fold, at least 10- fold, at least 15-fold, at least 20-fold, and at least 25-fold more thermostable than the parent when residual activity is compared using the assay for determining remaining activity (azo- casein) in the Examples.
• a protease variant can comprise various types of modifications relative to a template (i.e. a parent or reference protease, or a comparative amino acid sequence such as amino acids 1 to 177 of SEQ ID NO: 2): An amino acid can be substituted with another amino acid; an amino acid can be deleted; an amino acid can be inserted between two residues; as well as any combination of any number of such modifications.
• a template i.e. a parent or reference protease, or a comparative amino acid sequence such as amino acids 1 to 177 of SEQ ID NO: 2
• An amino acid can be substituted with another amino acid; an amino acid can be deleted; an amino acid can be inserted between two residues; as well as any combination of any number of such modifications.
• a substitution or extension without any indication of what to substitute or extend with refers to the insertion of any natural, or non-natural, amino acid, except the one that occupies this position in the template.
• a substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.
• insertion is intended to cover also N- and/or C-terminal extensions.
• the mature polypeptide comprised in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in the variant protease.
• the amino acid sequence of the variant protease is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.
• substitutions For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as "Thr226Ala” or “T226A”. Multiple mutations are separated by slash marks ("/"), e.g., "Gly205Arg / Ser41 1 Phe” or "G205R / S41 1 F", representing substitutions at positions 205 and 41 1 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.
• slash marks e.g., "Gly205Arg / Ser41 1 Phe” or "G205R / S41 1 F”
• Insertions For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated "Gly195Glyl_ys" or "G195GK".
• Variants comprising multiple alterations are separated by slash marks ("/"), e.g., "Arg170Tyr/Gly195Glu” or “R170Y/G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively.
• slash marks e.g., "Arg170Tyr/Gly195Glu” or "R170Y/G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively.
• amino acid positions are based on the amino acid sequence of the protease derived from Thermoascus aurantiacus CGMCC No. 0670, the mature polypeptide sequence of which is given in the sequence lisiting as amino acids 1 to 177 of SEQ ID NO: 2 (amino acids 1 -177 of SEQ ID NO:2). Accordingly, in the present context, the basis for numbering positions is SEQ ID NO:2 starting with T1 and ending with C177.
• the term "mature" part refers to that part of the polypeptide which is secreted by a cell which contains, as part of its genetic equipment, a polynucleotide encoding the polypeptide.
• the mature polypeptide part refers to that part of the polypeptide which remains after the signal peptide part, as well as a propeptide part, if any, has been cleaved off.
• the signal peptide part can be predicted by programs known in the art (e.g. SignalP). Amino acids 1 to 177 of SEQ ID NO: 2 is the expected mature part.
• the first amino acid of the mature part of an enzyme can be determined by N-terminal sequencing of the purified enzyme. Any difference between the signal peptide part and the mature part must then be due to to the presence of a propeptide.
• the present invention relates to methods of producing a protease variant having protease activity and an improved thermostablity when compared to the wild-type parent protease, said method comprising culturing a cell into which has been introduced an expression vector comprising the following operably linked elements:
• a polynucleotide molecule encoding a protease variant which has at least 75% identity to the parent protease shown in amino acids 1 to 177 of SEQ ID NO: 2 and which comprises at least one modification as compared to amino acids 1 to 177 of SEQ ID NO: 2 in at least one position selected from the following: 27, 79, 82, 87, 1 12, 142, 2, 5, 6, 8, 26, 41 , 43, 46, 49, 53, 54, 73, 88, 104, 1 14, 1 15, 1 16, 126, 152, 157, 158, and 173,
• polynucleotide molecule is prepared by introducing at least one mutation into a DNA molecule encoding a protease
• said cell expresses the protease variant encoded by the polynucleotide molecule; and recovering the protease variant.
• the protease applied in the method of the invention exhibiting improved thermostability comprises at least one of the following modifications: A27K, A27G, A27V, Q53K, Q53R, T54R, D79K, D79L, D79M, Y82F, S87G, S87P, A1 12P, D142L, R2P, AS5, C6R, ⁇ 8, N26R, S41 R, Y43F, T46R, S49R, A73C, P81 R, N88R, D104R, D104P, T1 14P, S1 15R, T1 16V, T124L, T124V, A126V, M152R, S157K, Q158W, and 1173V.
• the present invention relates to a protease variant which has at least 75% identity to amino acids 1 to 177 of SEQ I D NO: 2 and which comprises at least one modification as compared to amino acids 1 to 177 of SEQ ID NO: 2 in at least one position selected from the following: 27, 79, 82, 87, 1 12, 142, 2, 5, 6, 8, 26, 41 , 43, 46, 49, 53, 54, 73, 88, 104, 1 14, 1 15, 1 16, 126, 152, 157, 158, and 173.
• the protease variants comprise at least one modification in at least one position selected from the group consisting of positions: 27, 79, 82, 87, 104,1 12, 126, and 142.
• the protease of the invention exhibiting improved thermostability comprises at least one of the following modifications: A27K, A27G, A27V, Q53K, Q53R, T54R, D79K, D79L, D79M, Y82F, S87G, S87P, A1 12P, D142L, R2P, AS5, C6R, AG8, N26R, S41 R, Y43F, T46R, S49R, A73C, P81 R, N88R, D104P, D104R, T1 14P, S1 15R, T1 16V, T124L, T124V, A126V, M152R, S157K, Q158W, and 1173V.
• the position numbers refer to the position numbering of amino acids 1 to 177 of SEQ ID NO: 2, as described in the section "Position Numbering.” Positions corresponding to these amino acids 1 to 177 of SEQ ID NO: 2 position numbers in other proteases are determined as described above.
• the variant protease of the invention is a variant of the protease of amino acids 1 to 177 of SEQ ID NO: 2, viz. it is not identical to amino acids 1 to 177 of SEQ ID NO: 2, as it comprises at least one modification as compared to amino acids 1 to 177 of SEQ ID NO: 2.
• the protease comprises at least one of the following combinations of modifications:
• a homology model of the structure of SEQ ID NO: 2 was build using PDB entry 1 EB6 as a template.
• the model was build using the Yasara program (http://yasara.org. YASARA Biosciences, Neue-Welt-Hoehe 13/b, 8042 Graz, Austria / Europe).
• the model was subjected to molecular dynamics (MD) simulations, electrostatic calculations ancestral sequence reconstruction, consensus sequence calculation, and auto- cleavage site prediction. Based on the modelled structure and the simulation results, modifications were suggested with particular emphasis at improving the thermostability properties.
• MD molecular dynamics
• positions close to the active site Zn ion were identified.
• D142 is an example of a residue close to the active site.
• posions exposed to the surface were identified. Positions on the surface interact with the water and are important for stability. Positions 2, 5, 6, 8, 26, 27, 41 , 49, 53, 73, 79, 87, 88, 104, 1 12, 1 14, 1 15, 1 16, 157, 158 are exposed to the surface. The most important of these are 1 12, 79 and 87.
• Positions with charged amino acids in un-favorable positions include: 2, 19, 42, 52, 58, 60, 64, 80, 104, 121 , 143 and in particular 142 and 79.
• the loss of activity of the protease can be related to auto-proteolysis. Based on mass spec analysis of self-digested protein of SEQ ID NO: 2 combined with specificity data from homologous enzymes, a number of positions were suggested: 59, 100, 105, 130, 131 , 148 and in particular position 27.
• Prolines are known to affect stability, and using the model structure, positions compatible with substituting a proline were identified using the function SUGPRO in the program WHATIF (http://www.cmbi.kun.nl/whatif. WHAT IF Foundation / CMBI, Toernooiveld 1 , 6525 ED Nijmegen, The Netherlands).
• Cystein bridges can stabilise the structure and by applying the SUGCYS function of the WHATIF program on the model structure positions comparable with cystein substitutions were identified.
• protease variants were prepared by methods known in the art and tested as described in the experimental part.
• Whether or not a variant protease of the invention has an improved thermostability as compared to a parent protease may be determined as described in Example 1.
• the variant protease of the invention may have an improved thermostability as compared to a parent protease, wherein the improved thermostability is determined as increased relative activity.
• the variant protease of the invention may have an improved thermostability as compared to a parent protease, wherein the improved thermostability is determined as increased remaining activity.
• Thermostability may also be determined using DSC measurements to determine the denaturation temperature, Td, of the purified protease protein.
• Td is indicative of the thermostability of the protein: The higher the Td, the higher the thermostability.
• the protease of the invention has a Td which is higher than the Td of a reference protease, wherein Td is determined on purified protease samples after buffer- exchange to 20 mM Na-acetate pH 4.5 or 5.5 w/ or w/o 2.5 mM Zn 2+ by use of a centrifugal filter device (10,000 MWCO)(, using Differential Scanning Calorimetry at a 90°C/h scan rate from 20- 1 10°C (we have a few scans up to 120°C)in 20 mM Na-acetate buffer.
• a centrifugal filter device 10,000 MWCO
• the Td of the protease of the invention is higher than the Td of the protease of amino acids 1 to 177 of SEQ ID NO: 2, more preferably at least 101 % thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or at least 1 10% thereof. Even more preferably, the Td of the protease of the invention is at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% of the Td of the protease of amino acids 1 to 177 of SEQ ID NO: 2.
• thermostable protease of the invention has a melting temperature, Tm (or a denaturation temperature, Td), as determined using Differential Scanning Calorimetry (DSC) as described in the Examples (i.e. in 20 mM sodium acetate,), of at least 50°C.
• Tm melting temperature
• Td denaturation temperature
• the Tm is at least 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or at least 100°C.
• the invention relates to improved thermostability variants of the parent protease of amino acids 1 to 177 of SEQ ID NO: 2 comprising at least one of the following modifications: A27K, A27G, A27V, Q53K, Q53R, T54R, D79K, D79L, D79M, Y82F, S87P, S87G, A1 12P, D142L, R2P, AS5, C6R, ⁇ 8, N26R, S41 R, Y43F, T46R, S49R, A73C, P81 R, N88R, D104R, D104P, T1 14P, S1 15R, T1 16V, T124L, T124V, A126V, M152R, S157K, Q158W, and 1173V.
• the modifications are selected from the group consisting of: A27K, D79L, Y82F, S87G, S87P, D104P, A1 12P, A126V,
• the invention relates to improved thermostability variants of the parent protease of amino acids 1 to 177 of SEQ ID NO: 2 comprising modifications selected from:
• AS5/D79L/S87P AS5/D79L/S87P, AS5/D79L/S87P/A1 12P/D142L, AS5/N26R/D79L/S87P/A1 12P/D142L, C6R/D79L/S87P, AG8/D79L/S87P, N26R/D79L/S87P, N26R/T46R/D79L/S87P/A1 12P/D142L, A27G/D79L/S87P/A1 12P/D142L, A27K/D79L/S87P/A1 12P/D142L,
• A27K/D79L/S87P/A1 12P/T124V/D142L A27V/D79L/S87P/A1 12P/D142L
• S41 R/D79L/S87P S41 R/D79L/S87P/A1 12P/D142L
• S41 R/D79L/S87P/A1 12P/D142L/S157K S41 R/D79L/S87P/A1 12P/D142L/S157K
• the invention relates to an improved thermostability variant of the parent protease of amino acids 1 to 177 of SEQ ID NO: 2 comprising the modifications:
• the invention relates to an improved thermostability variant of the parent protease of amino acids 1 to 177 of SEQ ID NO: 2 comprising the modifications: A27K/Y82F/S87G/D104P/A1 12P/A126V/D142L.
• the invention relates to an improved thermostability variant of the parent protease of amino acids 1 to 177 of SEQ ID NO: 2 comprising the modifications: A27K/D79L/Y82F/D104P/A1 12P/A126V/D142L
• the invention relates to an improved thermostability variant of the parent protease of amino acids 1 to 177 of SEQ ID NO: 2 comprising the modifications: A27K/Y82F/D104P/A1 12P/A126V/D142L.
• the present invention also relates to nucleic acid sequences comprising a nucleic acid sequence which encodes a protease variant of the invention.
• isolated nucleic acid sequence refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis.
• an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced.
• the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated.
• the nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
• the nucleic acid sequences of the invention can be prepared by introducing at least one mutation into a template protease coding sequence or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a variant protease.
• the introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by any of the methods known in the art, e.g. by site-directed mutagenesis, by random mutagenesis, or by doped, spiked, or localized random mutagenesis.
• Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.
• the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be performed so that codons for unwanted amino acids are avoided.
• the doped or spiked oligonucleotide can be incorporated into the DNA encoding the protease enzyme by any technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
• the doping is carried out using "constant random doping", in which the percentage of wild-type and mutation in each position is predefined.
• the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues.
• the doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% mutations in each position.
• An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
• the random mutagenesis may be advantageously localized to a part of the parent protease in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme.
• a nucleic acid construct comprises a nucleic acid sequence of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
• Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
• nucleic acid construct refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature.
• nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
• control sequences is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention.
• Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide.
• control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, transcription promoter, signal peptide sequence, and transcription terminator.
• the control sequences include a transcription promoter, and transcriptional and translational stop signals.
• the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
• operably linked denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
• coding sequence means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product.
• the boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG.
• the coding sequence may a DNA, cDNA, or recombinant nucleotide sequence
• expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
• expression vector is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention, and which is operably linked to additional nucleotides that provide for its expression.
• a nucleic acid sequence encoding a protease variant of the invention can be expressed using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
• the recombinant expression vector carrying the DNA sequence encoding a protease variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
• the protease variant may also be co-expressed together with at least one other enzyme of interest, such as a glucoamylase, alpha-amylase, phosphatase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, and/or beta-glucanase.
• the enzymes may be co-expressed from different vectors, from one vector, or using a mixture of both techniques. When using different vectors, the vectors may have different selectable markers, and different origins of replication. When using only one vector, the genes can be expressed from one or more promoters.
• the protease variant may also be expressed as a fusion protein, i.e. that the gene encoding the protease variant has been fused in frame to the gene encoding another protein.
• This protein may be another enzyme or a functional domain from another enzyme.
• host cell includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct comprising a polynucleotide of the present invention.
• the present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides.
• a vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
• the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
• the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.
• Useful unicellular microorganisms are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus lichen iformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans and Streptomyces murinus, or gram negative bacteria such as E.
• a Bacillus cell e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagul
• the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.
• the Bacillus cell is an alkalophilic Bacillus.
• the introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 1 1 1-1 15), using competent cells (see, e.g., Young and Spizizin, 1961 , Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).
• protoplast transformation see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 1 1 1-1 15
• competent cells see, e.g., Young and Spizizin, 1961 , Journal of Bacteriology 81 :
• the host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
• the host cell is a fungal cell.
• "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171 ) and all mitosporic fungi (Hawksworth et al., 1995, supra).
• the fungal host cell is a yeast cell.
• yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
• the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
• the yeast host cell is a Pichia pastoris, Pichia methanolica, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
• the yeast host cell is a Kluyveromyces lactis cell.
• the yeast host cell is a Yarrowia lipolytica cell.
• the fungal host cell is a filamentous fungal cell.
• filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
• the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
• Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
• vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
• the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
• the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell.
• the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.
• the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koning
• Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proc.Nat.Acad.Sci. USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
• the present invention also relates to methods for producing a protease of the present invention comprising (a) cultivating a host cell; and (b) recovering the protease.
• the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art.
• the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
• the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
• the resulting polypeptide may be recovered using methods known in the art.
• the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
• polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, Eds, VCH Publishers, New York, 1989).
• chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
• electrophoretic procedures e.g., preparative isoelectric focusing
• differential solubility e.g., ammonium sulfate precipitation
• SDS-PAGE or extraction
• the present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleotide sequence encoding a polypeptide having protease activity of the present invention so as to express and produce the polypeptide in recoverable quantities.
• the polypeptide may be recovered from the plant or plant part.
• the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.
• the polypeptide is targeted to the endosperm storage vacuoles in seeds.
• This can be obtained by synthesizing it as a precursor with a suitable signal peptide, see Horvath et al in PNAS, Feb. 15, 2000, vol. 97, no. 4, p. 1914-1919.
• the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot) or engineered variants thereof.
• monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilized hybrid of wheat (Triticum) and rye (Secale), and maize (corn).
• dicot plants are tobacco, legumes, such as sunflower (Helianthus), cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
• Low-phytate plants as described e.g. in US patent no. 5,689,054 and US patent no. 6,1 1 1 ,168 are examples of engineered plants.
• plant parts are stem, callus, leaves, root, fruits, seeds, and tubers, as well as the individual tissues comprising these parts, e.g. epidermis, mesophyll, parenchyma, vascular tissues, meristems. Also specific plant cell compartments, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e.g. embryos, endosperms, aleurone and seed coats.
• the transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
• the expression construct is a nucleic acid construct which comprises a nucleic acid sequence encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleic acid sequence in the plant or plant part of choice.
• the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
• regulatory sequences such as promoter and terminator sequences and optionally signal or transit sequences are determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed.
• the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific cell compartment, tissue or plant part such as seeds or leaves.
• Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
• the following promoters may be used: The 35S-CaMV promoter (Franck et al., 1980, Cell 21 : 285-294), the maize ubiquitin 1 (Christensen AH, Sharrock RA and Quail 1992. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation), or the rice actin 1 promoter (Plant Mol. Biol. 18, 675-689.; Zhang W, McElroy D. and Wu R 1991 , Analysis of rice Act1 5' region activity in transgenic rice plants. Plant Cell 3, 1 155-1 165).
• Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol.
• a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708-71 1 ), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941 ), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772.
• a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889)
• the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
• the promoter may be inducible by abiotic treatments such as temperature, drought or modifications in salinity or inducible by exogenously applied substances that activate the promoter, e.g. ethanol, oestrogens, plant hormones like ethylene, abscisic acid, gibberellic acid, and/or heavy metals.
• abiotic treatments such as temperature, drought or modifications in salinity or inducible by exogenously applied substances that activate the promoter, e.g. ethanol, oestrogens, plant hormones like ethylene, abscisic acid, gibberellic acid, and/or heavy metals.
• a promoter enhancer element may also be used to achieve higher expression of the polypeptide in the plant.
• the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention.
• Xu et al., 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression.
• codon usage may be optimized for the plant species in question to improve expression (see Horvath et al referred to above).
• the selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
• the nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including / ⁇ grot»acien ' t/m-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
• ⁇ grobacfer/i/m-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), and it can also be used for transforming monocots, although other transformation methods are more often used for these plants.
• the method of choice for generating transgenic monocots, supplementing the Agrobacterium approach is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281 ; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674).
• An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21 : 415-428.
• the transformants having incorporated therein the expression construct are selected and regenerated into whole plants according to methods well-known in the art.
• the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using e.g. co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.
• the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a polypeptide having protease activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
• the present invention relates to compositions comprising a polypeptide of the present invention, as well as methods of using these.
• polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
• the polypeptide composition may be in the form of granulates or microgranulates.
• the polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.
• the polypeptide and/or composition of the invention may be used for a detergent composition, an animal feed composition, for starch liquefaction, saccharification, and/or fermentation, e.g., as disclosed in WO9220777.
• the polypeptide and/or composition of the invention may be used in a process for production of a syrup.
• the end product may be glucose, but may also be converted, e.g., by glucose isomerase to fructose or a mixture composed almost equally of glucose and fructose.
• This mixture, or a mixture further enriched with fructose is the most commonly used high fructose corn syrup (HFCS) commercialized throughout the world.
• HFCS high fructose corn syrup
• the polypeptide and/or composition of the invention may be used in a process for production of a fermentation product.
• the invention relates to a method for producing a fermentation product, comprising (a) fermenting using a fermenting microorganism, and a carbohydrate containing material in the presence of a polypeptide having protease activity of any of claims 6 to 10, and (b) producing the fermentation product from the fermented carbohydrate containing material.
• Fermentation products include alcohols (e.g., ethanol, methanol, butanol, 1 ,3- propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H 2 and C0 2 ), and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 , beta-carotene); hormones, and other compounds which are difficult to produce synthetically.
• organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid
• Fermentation processes are also commonly used to produce consumable alcohol (e.g., beer and wine).
• a preferred fermentation product is ethanol, e.g., industrial ethanol, fuel ethanol and/or potable ethanol.
• Other preferred fermentation products include the co-products from the ethanol fermentation process, e.g., distillers dried grain (DDG).
• DDG distillers dried grain
• starch is liquefied in the presence of an alpha-amylase
• the liquefied mash is saccharified in the presence of a glucoamylase.
• the saccharified mash is fermented with a yeast; and (d) the ethanol produced is recovered.
• a polypeptide of the invention is added to the mash prior to/during liquefaction, or saccharification and/or to the hydrolysed starch and sugars during the fermentation.
• compositions comprising a polypeptide of the present invention comprises at least one other polypeptide selected from amongst amylase such as, for example, alpha- amylase (EC 3.2.1.1 ), glucoamylase (EC 3.2.1.3) and pullulanase (EC 3.2.1.41 ); phytase (EC 3.1 .3.8 or 3.1 .3.26); phosphatase (EC 3.1 .3.1 ; EC 3.1.3.2; EC 3.1 .3.39); xylanase (EC 3.2.1 .8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.-.- ), phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1 .1.5); phospholipase C (3.1.4.3); phospholipast amy
• Chemicals used were commercial products of at least reagent grade.
• E.coli DH12S available from Gibco BRL was used for yeast plasmid rescue.
• pJTPOOO is a S. cerevisiae and E.coli shuttle vector under the control of TPI promoter, constructed from pJC039 described in WO 01/92502, in which the Thermoascus aurantiacus M35 protease gene (WO 03048353) has been inserted.
• Saccharomyces cerevisiae YNG318 competent cells MATa Dpep4[cir+] ura3-52, Ieu2- D2, his 4-539 was used for protease variants expression. It is described in J. Biol. Chem. 272 (15), pp 9720-9727, 1997.
• the solution is sterilized using a filter of a pore size of 0.20 micrometer.
• Agar (2%) and H 2 0 (approx. 761 ml) is autoclaved together, and the separately sterilized SC-glucose solution is added to the agar solution.
• YPD Bacto peptone 20 g/l, yeast extract 10 g/l, 20 % glucose 100 ml/l.
• YPD+Zn YPD+0.25 mM ZnS0 4.
• PEG/LiAc solution 40 % PEG4000 50 ml, 5 M Lithium Acetate 1 ml.
• Each well contains 200 microL of 0.05-0.1 % of zein (Sigma), 0.25 mM ZnS0 4 and 1 % of agar in 20 mM sodium acetate buffer, pH 4.5.
• zein Sigma
• 0.25 mM ZnS0 4 0.25 mM ZnS0 4
• 1 % of agar in 20 mM sodium acetate buffer, pH 4.5.
• Yeast transformation was performed using the lithium acetate method. 0.5 microL of vector (digested by restriction endnucleases) and 1 microL of PCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competent cells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a 12 ml polypropylene tube (Falcon 2059). Add 0.6 ml PEG/LiAc solution and mix gently. Incubate for 30 min at 30 °C, and 200 rpm followed by 30 min at 42 °C (heat shock). Transfer to an eppendorf tube and centrifuge for 5 sec. Remove the supernatant and resolve in 3 ml of YPD.
• E.coli transformation for DNA sequencing was carried out by electroporation (BIO-RAD Gene Pulser).
• DNA Plasmids were prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or with the Qiagen® Plasmid Kit. DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNA Engine. The ABI PRISMTM 310 Genetic Analyzer was used for determination of all DNA sequences.
• Themoascus M35 protease gene was amplified with the primer pair Prot F (SEQ ID NO: 3) and Prot R (SEQ ID NO: 4).
• the resulting PCR fragments were introduced into S. cerevisiae YNG318 together with the pJC039 vector (described in WO2001/92502) digested with restriction enzymes to remove the Humicola insolens cutinase gene.
• Plasmid in yeast clones on SC-glucose plates was recovered to confirm the internal sequence and termed as pJTPOOI .
• the primers AM34 (SEQ ID NO:5) and AM35 (SEQ ID NO:6) were used to make DNA fragments containing any mutated fragments by the SOE method together with degenerated primers (AM34 + Reverse primer and AM35 + forward primer) or just to amplify a whole protease gene (AM34 + AM35).
• DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. The resulting purified fragments were mixed with the vector digest. The mixed solution was introduced into Saccharomyces cerevisiae to construct libraries or site-directed variants by in vivo recombination.
• Yeast clones on SC-glucose were inoculated to a well of a 96-well micro titre plate containing YPD+Zn medium and cultivated at 28 °C for 3 days.
• the culture supernatants were applied to a 96-well zein micro titer plate and incubated at at least 2 temperatures (ex. 60 °C and 65 °C, 70 °C and 75 °C, 70 °C and 80 °C) for more than 4 hours or overnight.
• the turbidity of zein in the plate was measured as A630 and the relative activity (higher/lower temperatures) was determined as an indicator of thermoactivity improvement.
• the clones with higher relative activity than the parental variant were selected and the sequence was determined.
• Yeast clones on SC-glucose were inoculated to a well of a 96-well micro titre plate and cultivated at 28 °C for 3 days.
• Protease activity was measured at 65 °C using azo-casein (Megazyme) after incubating the culture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 min at a certain temperature (80 °C or 84 °C with 4 °C as a reference) to determine the remaining activity.
• the clones with higher remaining activity than the parental variant were selected and the sequence was determined.
• TCA trichloroacetic acid
• the constructs comprising the protease variant genes were used to construct expression vectors for Aspergillus.
• the Aspergillus expression vectors consist of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the plasmid was the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source.
• the expression plasmids for protease variants were transformed into Aspergillus as described in Lassen et al. (2001 ), Appl. Environ. Microbiol. 67, 4701-4707. For each of the constructs 10- 20 strains were isolated, purified and cultivated in shake flasks.
• fractions are assayed by an endo-protease assay (cf. below) followed by standard SDS-PAGE (reducing conditions) on selected fractions. Fractions are pooled based on the endo-protease assay and SDS-PAGE.
• Protazyme OL tablet/5 ml 250 mM Na-acetate pH 5.0 is dissolved by magnetic stirring (substrate: endo-protease Protazyme AK tablet from Megazyme - cat. # PRAK 1 1/08).
• the tubes are incubated on a Thermomixer with shaking (1000 rpm) at 50°C for 15 minutes.
• Zein-BCA assay was performed to detect soluble protein quantification released from zein by variant proteases at various temperatures.

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